The present invention is a system for the design of diagnostics having an amplified or differential response as a result of the inclusion of a ribozyme.
The detection of infectious agents or molecules indicative of disease, including metabolites, nucleic acids, and proteins, is a fundamental component in the diagnosis and treatment of medical disorders, as well as in research. A number of methodologies are currently in use for detection. These methodologies can generally be divided into antibody-based diagnostic assays for proteins, either components of the disease causing agent or byproducts of the disease, and diagnostic assays for nucleic acids, such as the genetic material encoding a component of the disease causing agent.
Assays for proteins are further divided into those methods which involve a binding reaction between a molecule (usually an antibody) and the protein to be detected, or a reaction between an enzyme which binds the targeted molecule resulting in activation of the enzyme so that it can cleave a substrate to produce a detectable color change. Many of the binding assays include a dye, an enzyme, or a radioactive or fluorescent label to enhance detection. Antibodies to the protein can be obtained from patients, immunized animals, or antigen-specific monoclonal cell lines. These antibody assays include assays such as sandwich ELISA assays, Western immunoblot, radioimmunoassays, and immunodiffusion assays. Other assays use molecules such as avidin and biotin for immobilization and detection of molecules. Techniques for preparing these reagents and methods for use thereof are known to those skilled in the art.
Assays for nucleic acid sequences range from simple methods for detection, such as a Northern blot hybridization using a radiolabeled probe to detect the presence of a nucleic acid molecule, to the use of the polymerase chain reaction (PCR) to amplify a very small quantity of a specific nucleic acid sequence to the point at which it can be used for detection of the sequence by hybridization techniques. Nucleotide probes can be labelled using dyes, or enzymatic, fluorescent, chemiluminescent, or radioactive labels which are commercially available. These probes can be used to detect by hybridization the expression of a gene or related sequences in cells or tissue samples in which the gene is a normal component, as well as to screen sera or tissue samples from humans suspected of having a disorder arising from infection with an organism, or to detect novel or altered genes as might be found in tumorigenic cells. Nucleic acid primers can also be prepared which, with reverse transcriptase or DNA polymerase and the polymerase chain reaction, can be used for detection of nucleic acid molecules which are present in very small amounts in tissues or fluids.
Only the enzyme-based methodologies and PCR (which uses a polymerase) are inherently catalytic, with detection linked to amplification of the signal. PCR has several disadvantages, although it is capable of detecting very small quantities of DNA: it requires a high degree of technical competence for reliability; it is extremely sensitive to contamination resulting in false positives; it is difficult to use quantitatively rather than qualitatively. The other methods rely on conjugation of an enzyme, usually to additional components of the assay, to increase signal generation and amplification. The use of these additional ligands increases the noise of the system, with higher background and false positives, and necessitates several levels of control reactions.
Ribozymes are defined as RNA molecules having enzyme like activity. There are three general pathways of RNA catalyzed cleavage: (1) cleavage by viroid-like RNA; (2) cleavage by RNAase P or the RNA component of RNAase P, the work of Sidney Altman at Yale University; and (3) cleavage by the Tetrahymena ribozyme, the work of Thomas Cech at the University of Colorado. All naturally occurring ribozymes known to date, with the exception of RNAase P, work in cis and must be engineered to work in trans, i.e., on another molecule. This is accomplished by separating the portion of the RNA molecule with enzymatic activity from the portion serving as substrate, and conferring substrate-like properties, including appropriate secondary and tertiary structure, on the target molecule which is to be cleaved. Specificity can be conferred by adding complementary nucleic acid sequence which hybridize adjacent to the site to be cleaved on the target molecule.
Each class of ribozyme cleaves a different sequence of nucleotides using distinct mechanisms of action. Moreover, each class is further distinguished based on how many nucleotide bases are essential for enzymatic activity and to the extent the intended target and the ribozyme can be manipulated to alter specificity.
The Tetrahymena ribozyme was the first ribozyme to be discovered. This ribozyme is guanosine-dependent for its cleavage. It is a large ribozyme that naturally operates in cis. A smaller internal portion can be engineered to operate in trans, that is, on a separate molecule, targeting specific four nucleotide sequences.
M1 RNA, the RNA ribozyme subunit of E. coli RNAase P, is a nearly 400-base RNA molecule which cleaves a whole variety of separate, other molecules in the cell to produce mature tRNAs from their precursors. Other molecules can be converted into substrate for M1 RNA through the use of an external guide sequence characterized as an isolated oligoribonucleotide having at its 5' terminus at least seven nucleotides complementary to the nucleotides 3' to the cleavage site in the RNA to be cleaved and at its 3' terminus the nucleotides N C C A directly joined to the complementary nucleotides, wherein N is any nucleotide and the complementary nucleotides in the oligoribonucleotide hybridizes to the complementary nucleotides in the RNA to be cleaved, as described by Forster and Altman, in Science 249:783-786 (1990), "External Guide Sequences for an RNA Enzyme". Altman, et al., Proc. Natl. Acad. Sci. 89 (17):8006-8010 (1992), "Targeted Cleavage of Messenger RNA by Human RNase P" recently described the construction of an external guide sequence for the eukaryotic equivalent of the E. coli RNAase P, based on a structure derived from a precursor TRNA. The RNAase P reaction and the Tetrahymena reaction both act by creating 5'-phosphate and 3'-hydroxyl termini.
There are several kinds of viroid-like RNA ribozymes found in plants and animals. The hammerhead ribozyme is one class in this category and the hepatitis delta ribozyme is a second class. Unlike the RNAase P and Tetrahymena ribozymes, the engineered trans-acting plant viroid-like ribozymes are only 18 to 20 nucleotide bases long with equally short substrates. The central motif is a characteristic conserved sequence motif that Uhlenbeck demonstrated in 1987 and published in Nature 328:596-600 (1987), in which he proposed that all hammerheads shared certain features. The cleavage reaction of the viroid-like ribozymes creates a 2', 3' cyclic phosphate and a 5' hydroxyl terminus. Accordingly, there is a fundamentally and mechanistically different chemistry for these viroid-like RNA reactions as contrasted with the M1 RNA reaction or the Tetrahymena reaction. However, the end result is the same, i.e., cleavage of a separate RNA molecule.
It has been proposed by several groups that ribozymes have the potential to be used to treat disease or genetic disorders by cleaving target RNA, such as viral RNA or MRNA transcribed from genes which should be, but are not, turned off. No one has proposed using them as diagnostics, however.
It is therefore an object of the present invention to provide a means for amplification or generation of a diagnostic response using ribozymes.
It is a further object of the present invention to utilize ribozymes to serve as a catalyst in the detection of nucleic acids, proteins, and other molecules, in which detection is a result of cleavage of a substrate by the ribozyme.